Renato N. Soriano

Involvement of l‐glutamate and ATP in the neurotransmission of the sympathoexcitatory component of the chemoreflex in the commissural nucleus tractus solitarii of awake rats and in the working heart–brainstem preparation

Peripheral chemoreflex activation with potassium cyanide (KCN) in awake rats or in the working heart–brainstem preparation (WHBP) produces: (a) a sympathoexcitatory/pressor response; (b) bradycardia; and (c) an increase in the frequency of breathing. Our main aim was to evaluate neurotransmitters involved in mediating the sympathoexcitatory component of the chemoreflex within the nucleus tractus solitarii (NTS). In previous studies in conscious rats, the reflex bradycardia, but not the pressor response, was reduced by antagonism of either ionotropic glutamate or purinergic P2 receptors within the NTS. In the present study we evaluated a possible dual role of both P2 and NMDA receptors in the NTS for processing the sympathoexcitatory component (pressor response) of the chemoreflex in awake rats as well as in the WHBP. Simultaneous blockade of ionotropic glutamate receptors and P2 receptors by sequential microinjections of kynurenic acid (KYN, 2 nmol (50 nl)−1) and pyridoxalphosphate‐6‐azophenyl‐2′,4′‐disulphonate (PPADS, 0.25 nmol (50 nl)−1) into the commissural NTS in awake rats produced a significant reduction in both the pressor (+38 ± 3 versus+8 ± 3 mmHg) and bradycardic responses (−172 ± 18 versus−16 ± 13 beats min−1; n= 13), but no significant changes in the tachypnoea measured using plethysmography (270 ± 30 versus 240 ± 21 cycles min−1, n= 7) following chemoreflex activation in awake rats. Control microinjections of saline produced no significant changes in these reflex responses. In WHBP, microinjection of KYN (2 nmol (20 nl)−1) and PPADS (1.6 nmol (20 nl)−1) into the commissural NTS attenuated significantly both the increase in thoracic sympathetic activity (+52 ± 2%versus+17 ± 1%) and the bradycardic response (−151 ± 17 versus−21 ± 3 beats min−1) but produced no significant changes in the increase of the frequency of phrenic nerve discharge (+0.24 ± 0.02 versus+0.20 ± 0.02 Hz). The data indicate that combined microinjections of PPADS and KYN into the commissural NTS in both awake rats and the WHBP are required to produce a significant reduction in the sympathoexcitatory response (pressor response) to peripheral chemoreflex activation. We conclude that glutamatergic and purinergic mechanisms are part of the complex neurotransmission system of the sympathoexcitatory component of the chemoreflex at the level of the commissural NTS.

Sympathoexcitatory response to peripheral chemoreflex activation is enhanced in juvenile rats exposed to chronic intermittent hypoxia

In the present study, we tested the hypothesis that chronic intermittent hypoxia (CIH) produces changes in the autonomic and respiratory responses to acute peripheral chemoreflex activation. To attain this goal, 3‐week‐old rats were exposed to 10 days of CIH (6% O2 for 40 s at 9 min intervals; 8 h day−1). They were then used to obtain a working heart–brainstem preparation and, using this unanaesthetized experimental preparation, the chemoreflex was activated with potassium cyanide (0.05%, injected via the perfusion system), and the thoracic sympathetic nerve activity (tSNA), heart rate and phrenic nerve discharge (PND) were recorded. Rats subjected to CIH (n= 12), when compared with control animals (n= 12), presented the following significant changes in response to chemoreflex activation: (a) an increase in tSNA (78 ± 4 versus 48 ± 3%); (b) a long‐lasting increase in the frequency of the PND at 20 (0.52 ± 0.03 versus 0.36 ± 0.03 Hz) and 30 s (0.40 ± 0.02 versus 0.31 ± 0.02 Hz) after the stimulus; and (c) a greater bradycardic response (−218 ± 20 versus−163 ± 16 beats min−1). These results indicate that the autonomic and respiratory responses to chemoreflex activation in juvenile rats previously submitted to CIH are greatly increased.

Hydrogen sulfide inhibits preoptic prostaglandin E2 production during endotoxemia

Hydrogen sulfide (H(2)S) is a gaseous neuromodulator endogenously produced in the brain by the enzyme cystathionine β-synthase (CBS). We tested the hypothesis that H(2)S acts within the anteroventral preoptic region of the hypothalamus (AVPO) modulating the production of prostaglandin (PG) E(2) (the proximal mediator of fever) and cyclic AMP (cAMP). To this end, we recorded deep body temperature (Tb) of rats before and after pharmacological modulation of the CBS-H(2)S system combined or not with lipopolysaccharide (LPS) exposure, and measured the levels of H(2)S, cAMP, and PGE(2) in the AVPO during systemic inflammation. Intracerebroventricular (icv) microinjection of aminooxyacetate (AOA, a CBS inhibitor; 100 pmol) did not affect basal PGE(2) production and Tb, but enhanced LPS-induced PGE(2) production and fever, indicating that endogenous H(2)S plays an antipyretic role. In agreement, icv microinjection of a H(2)S donor (Na(2)S; 260 nmol) reduced the LPS-induced PGE(2) production and fever. Interestingly, we observed that the AVPO levels of H(2)S were decreased following the immunoinflammatory challenge. Furthermore, fever was associated with decreased levels of AVPO cAMP and increased levels of AVPO PGE(2). The LPS-induced decreased levels of cAMP were reduced to a lesser extent by the H(2)S donor. The LPS-induced PGE(2) production was potentiated by AOA (the CBS inhibitor) and inhibited by the H(2)S donor. Our data are consistent with the notion that the gaseous messenger H(2)S synthesis is downregulated during endotoxemia favoring PGE(2) synthesis and lowering cAMP levels in the preoptic hypothalamus.

Gaseous Mediators in Temperature Regulation

Deep body temperature (Tb) is kept relatively constant despite a wide range of ambient temperature variation. Nevertheless, in particular situations it is beneficial to decrease or to increase Tb in a regulated manner. Under hypoxia for instance a regulated drop in Tb (anapyrexia) is key to reduce oxygen demand of tissues when oxygen availability is diminished, leading to an increased survival rate in a number of species when experiencing low levels of inspired oxygen. On the other hand, a regulated rise in Tb (fever) assists the healing process. These regulated changes in Tb are mediated by the brain, where afferent signals converge and the most important regions for the control of Tb are found. The brain (particularly some hypothalamic structures located in the preoptic area) modulates efferent activities that cause changes in heat production (modulating brown adipose tissue activity and perfusion, for instance) and heat loss (modulating tail skin vasculature blood flow, for instance). This review highlights key advances about the role of the gaseous neuromodulators nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H2S) in thermoregulation, acting both on the brain and the periphery.

Role of hydrogen sulfide in the formalin-induced orofacial pain in rats.

Hydrogen sulfide (H2S) is a gasotransmitter synthesized in peripheral tissues by the enzyme cystathionine gamma-lyase (CSE). This gas has been documented to be involved in a wide variety of processes including inflammation and nociception. The aim of the present study was to investigate the role of the peripheral H2S pathway in nociceptive response to the orofacial formalin experimental model of pain. Orofacial pain was induced by subcutaneous injection of formalin (1.5%, 50 µl) into the upper lip of rats, and the time spent rubbing the face was measured at 3-min intervals for 45 min. Formalin induced a marked biphasic pain (first phase: 0-3 min; second phase: 15-33 min). Pretreatment with H2S donor (Na2S; 90 µmol/kg), CSE inhibitor (propargylglycine; 26.5 and 88.4 µmol/kg), or a preferential blocker of T-type Ca(2+) channels (mibefradil; 0.28 and 2.81 µmol/kg) attenuated the second phase of face rubbing when injected locally as well as systemically. Pretreatment with a selective blocker of K(+)ATP channels (glybenclamide; 2.81 µmol/kg) suppressed the Na2S-mediated attenuation of the formalin-induced pain second phase. Taken together these results suggest that endogenously produced H2S plays a pronociceptive role probably via T-type Ca(2+) channels, whereas exogenous H2S exerts antinociceptive effects mediated by K(+)ATP channels.

Reduced stress fever is accompanied by increased glucocorticoids and reduced PGE2 in adult rats exposed to endotoxin as neonates

Immune challenges during neonatal period may permanently program immune responses later in life, including endotoxin fever. We tested the hypothesis that neonatal endotoxin exposure affects stress fever in adult rats. In control rats (treated with saline as neonates; nSal) body temperature peaked approximately 1.5 degrees C during open-field stress, whereas in rats exposed to endotoxin (lipopolysaccharide, LPS) as neonates (nLPS) stress fever was significantly attenuated. Following stress, plasma corticosterone levels significantly increased from 74.29+/-7.05 ng ml(-1) to 226.29+/-9.87 ng ml(-1) in nSal rats, and from 83.43+/-10.31 ng ml(-1) to 324.7+/-36.87 ng ml(-1) in nLPS rats. Animals treated with LPS as neonates and adrenalectomized one week before experimentation no longer displayed the attenuated febrile response to stress. This attenuated stress fever caused by an increased corticosterone secretion is likely to be linked to an inhibitory effect of glucocorticoids on cyclooxygenase activity/PGE(2) production in preoptic/anteroventral third ventricular region (AV3V) since stress failed to cause a significant increase in PGE(2) in nLPS rats, and this effect was reverted by adrenalectomy. Altogether, the present results indicate that endogenous glucocorticoids are key modulators of the attenuated stress fever in adult rats treated with LPS as neonates, and they act downregulating PGE(2) production in AV3V. Moreover, our findings also support the notion that neonatal immune stimulus affects programming of stress responses during adulthood, despite the fact that inflammation and stress are two distinct processes mediated largely by different neurobiological mechanisms.

Exogenous ghrelin attenuates endotoxin fever in rats

Ghrelin is a gut-derived peptide that plays a role in energy homeostasis. Recent studies have implicated ghrelin in systemic inflammation, showing increased plasma ghrelin levels after endotoxin (lipopolysaccharide, LPS) administration. The aims of this study were (1) to test the hypothesis that ghrelin administration affects LPS-induced fever; and (2) to assess the putative effects of ghrelin on plasma corticosterone secretion and preoptic region prostaglandin (PG) E(2) levels in euthermic and febrile rats. Rats were implanted with a temperature datalogger capsule in the peritoneal cavity to record body core temperature. One week later, they were challenged with LPS (50 μg/kg, intraperitoneal, i.p.) alone or combined with ghrelin (0.1mg/kg, i.p.). In another group of rats, plasma corticosterone and preoptic region PGE(2) levels were measured 2h after injections. In euthermic animals, systemic administration of ghrelin failed to elicit any thermoregulatory effect, and caused no significant changes in basal plasma corticosterone and preoptic region PGE(2) levels. LPS caused a typical febrile response, accompanied by increased plasma corticosterone and preoptic PGE(2) levels. When LPS administration was combined with ghrelin fever was attenuated, corticosterone secretion further increased, and the elevated preoptic PGE(2) levels were relatively reduced, but a correlation between these two variables (corticosterone and PGE(2)) failed to exist. The present data add ghrelin to the neurochemical milieu controlling the immune/thermoregulatory system acting as an antipyretic molecule. Moreover, our findings also support the notion that ghrelin attenuates fever by means of a direct effect of the peptide reducing PGE(2) production in the preoptic region.

ACTIVATION OF PERIPHERAL CHEMORECEPTORS CAUSES POSITIVE INOTROPIC EFFECTS IN A WORKING HEART–BRAINSTEM PREPARATION OF THE RAT

1 The aim of the present study was to evaluate the effects of peripheral chemoreceptor activation on myocardial contractility in an anaesthetic‐free decerebrated rat preparation. 2 In the decerebrated and retrogradely perfused working heart–brainstem preparation, we recorded phrenic nerve activity, left ventricular (LV) pressure (microtip Millar catheter), LV dP/dT, heart rate and aortic perfusion pressure before and after activating peripheral chemoreceptors with bolus intra‐arterial injections of KCN. 3 Without cardiac pacing, chemoreflex activation caused falls in heart rate (–108 ± 21 b.p.m.) and complex polyphasic changes in LV pressure and LV dP/dT. If the heart was paced, chemoreflex activation caused significant rises in LP pressure (+16 ± 3 mmHg) and LV dP/dt (+778 ± 93 mmHg/s). These positive inotropic effects were significantly and substantially attenuated by β‐adrenoceptor blockade with atenolol. In all instances, chemoreflex activation elicited potent tachypnoeic responses. 4 In conclusion, activation of peripheral chemoreceptors in non‐anaesthetized rats evokes a positive inotropic response that is sympathetically mediated. This observation may be relevant for the evaluation of neurally induced effects of acute hypoxia on the ventricular myocardium.

INTERACTION BETWEEN THE CARBON MONOXIDE AND NITRIC OXIDE PATHWAYS IN THE LOCUS COERULEUS DURING FEVER

We have documented that the locus coeruleus (LC), the main noradrenergic nucleus in the brain, is part of a thermoeffector neuronal pathway in fever induced by lipopolysaccharide (LPS). Following this pioneering study, we have investigated the role of the LC carbon monoxide (CO) and nitric oxide (NO) pathways in fever. Interestingly, despite both CO and NO are capable of activating the same intracellular target, soluble guanylate cyclase (sGC), our data have shown that LC CO is an antipyretic molecule, whereas LC NO is propyretic. Thus, aiming at further exploring the mechanisms underlying their anti- and propyretic properties, we investigated the putative interplay between the LC CO and NO pathways. Male Wistar rats were implanted with a guide cannula in the fourth ventricle (4V) and a temperature datalogger capsule in the peritoneal cavity. The animals were microinjected into the 4V with an inhibitor of heme oxygenase (HO) (ZnDPBG [zinc(II)deuteroporphyrin IX 2,4 bis ethylene glycol]), or a CO donor (CORM-2 [tricarbonyldichlororuthenium-(II)-dimer]), or an inhibitor of nitric oxide synthase (NOS) (l-NMMA [N(G)-monomethyl-L-arginine acetate]), or an NO donor (NOC12 [3-ethyl-3-(ethylaminoethyl)-1-hydroxy-2-oxo-1-triazene]), and injected with LPS (100 μg/kg i.p.). Two hours later, the rats were decapitated, and the brains were frozen and cut in a cryostat. LC punches were processed to assess LC bilirubin and nitrite/nitrate (NOx) levels. Microinjection of ZnDPBG reduced LC bilirubin and increased LC NOx, whereas l-NMMA diminished LC NOx and reduced LC bilirubin. Furthermore, NOC12 caused an increase in LC bilirubin, whereas CORM-2 caused a reduction in LC NOx. These findings are consistent with the notion that in the LC during LPS fever the CO pathway downmodulates NOS activity and the NO pathway upmodulates HO activity, and, together with previous data, allow us to conjecture that LC CO blunts fever by downmodulating NOS (antipyretic property), LC NO upmodulates HO and sGC activities favoring the development of LPS fever (propyretic effect).

Propyretic role of the locus coeruleus nitric oxide pathway

Nitric oxide has been reported to modulate fever in the brain. However, the sites where NO exerts this modulation remain somewhat unclear. Locus coeruleus (LC) neurons express not only nitric oxide synthase (NOS) but also soluble guanylyl cyclase (sGC). In the present study, we evaluated in vivo and ex vivo the putative role of the LC NO–cGMP pathway in fever. To this end, deep body temperature was measured before and after pharmacological modulations of the pathway. Moreover, nitrite/nitrate (NOx) and cGMP levels in the LC were assessed. Conscious rats were microinjected within the LC with a non‐selective NOS inhibitor (NG‐monomethyl‐l‐arginine acetate), a NO donor (NOC12), a sGC inhibitor (1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one) or a cGMP analogue (8‐bromo‐cGMP) and injected intraperitoneally with endotoxin. Inhibition of NOS or sGC before endotoxin injection significantly increased the latency to the onset of fever. During the course of fever, inhibition of NOS or sGC attenuated the febrile response, whereas microinjection of NOC12 or 8‐bromo‐cGMP increased the response. These findings indicate that the LC NO–cGMP pathway plays a propyretic role. Furthermore, we observed a significant increase in NOx and cGMP levels, indicating that the febrile response to endotoxin is accompanied by stimulation of the NO–cGMP pathway in the LC.